Agarose gel electrophoresis is used to separate DNA fragments by size. DNA has a negative charge and will migrate toward the positive electrode in an electrical field. Shorter DNA fragments move faster through an agarose gel than longer fragments. Samples are run alongside a DNA ladder to determine the size of unknown DNA fragments. After electrophoresis, gels are stained with ethidium bromide and visualized under UV light to see DNA band patterns. This allows analysis of results such as identifying the presence of specific genes.

1. Agarose gel electrophoresis
by Dr Yousif H.M.Sharif
College of Dentistry /UOD
13th
practical Microbiology
11 April 2017

2. Why electrophoresis?
• To separate DNA
fragments from each
other
• To determine the sizes
of DNA fragments
• To determine the
presence or amount of
DNA
• To analyze restriction
digestion products

3. Background on Agarose Gel
Electrophoresis
• Agarose gel electrophoresis is a molecular
method to separate DNA or RNA molecules by
size.
• This is achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix with an electric field (electrophoresis).
• Shorter molecules move faster and migrate
faster than longer ones .

4. • DNA is negatively charged.
+-
Power
DNA
• When placed in an electrical field, DNA will migrate toward the
positive pole (anode).
H

O2

• An agarose gel is used to slow the movement of DNA and separate
by size.

5. +-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
small
large

6. Material required for agarose gel
electrophoresis
 Electrophoresis chamber
 Agarose gel
 Gel casting tray
 Running Buffer
 Staining agent (dye)
 A comb
 DNA ladder
 Sample to be separate

7. Casting tray
Gel combs
Power supply
Gel tank Cover
Electrical leads

Electrophoresis Equipment

8. Agarose
What is Agarose ?
Agarose is a linear polymer extracted from seaweed.
D-galactose 3,6-anhydro
L-galactose

9. Which running buffer Should I
use ?
• TBE (Tris-borate-EDTA) 1X if you look for small DNA
fragment ,Nice band
• TAE (Tris-acetate EDTA) 1X as running buffer –Large
DNA fragment ,less prone to overheat

10. %Percent agarose
•Most agarose gels are made with between 0.7% (good
separation or resolution of large 5–10kb DNA
fragments) and 2% (good resolution for small 0.2–1kb
fragments) agarose dissolved in electrophoresis buffer.
•1 %
Running Buffers
The most common being: Tris/Acetate/EDTA (TAE)
•Tris-acid solutions are effective buffers for slightly
basic conditions, which keep DNA deprotonated and
soluble in water.
•the role of the EDTA is to protect the nucleic acids
against enzymatic degradation

11. Preparing a 2% W/V Agarose Gel
• 2% W/V agarose = 2 grams agarose dissolved in 100
milliliters (mL) of Buffer
• For our purposes each gel tray requires 25 mLs of gel
• To prepare 25 ml of a 2% agarose gel
2 grams = X grams
100mls 25 mls
X = 0.5 grams agarose
• Weight 0.5 grams agarose and dissolve in 25 mls
buffer

12. Procedure of making agarose gel
• An agarose gel is prepared by combining
agarose powder and a buffer solution.

13. Agarose
Buffer
Flask for boiling
1.Make a 1% agarose solution in 20ml TAE.
（ 0.2g agarose in 20ml TAE OR TBE ）
2. Carefully bring the solution just to the boil
to dissolve the agarose.
3. Let the solution cool down to about 60 °C at
room temperature. Stir or swirl the solution
while cooling.
4.After complete dissolve and let it cool
around 40C, we simply add 2microliter of safe-
Dye then pour in to gel tray
STEPS

14. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is several times
larger than the volume of buffer.

15. Gel casting tray & combs

16. Seal the edges of the casting tray and put in the combs. Place the casting tray on a
level surface. None of the gel combs should be touching the surface of the casting
tray.
Preparing the Casting Tray

17. Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose to
dissolve.
***Be careful when boiling - the agarose solution may become superheated and may boil
violently if it has been heated too long in a microwave oven.
Melting the Agarose

18. Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the
melted agarose solution into the casting tray. Avoid air bubbles.
Pouring the gel

19. Each of the gel combs should be submerged in the melted agarose solution.

20. When cooled, the agarose polymerizes, forming a flexible gel. It should appear
lighter in color when completely cooled (30-45 minutes). Carefully remove the
combs and tape.

21. Place the gel in the electrophoresis chamber.

22. buffer 
Add enough electrophoresis buffer to cover the gel to a depth of at
least 1 mm. Make sure each well is filled with buffer.
Cathode
(negative)
Anode
(positive)

wells
  
DNA

23. Loading DYE: 
• Bromophenol Blue (for color)
• Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with Loading dye. This allows the samples to be seen
when loading onto the gel, and increases the density of the samples, causing
them to sink into the gel wells.

24. Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample. The sample
should sink into the well. Be careful not to puncture the gel with the pipette tip.

25. Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
Running the Gel

26.  wells
 Bromophenol Blue
Cathode
(-)
Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA
(-)


27.  100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
 12,000 bp
 5,000
 2,000
DNA Ladder Standard
DNA ladder (DNAs of known sizes) on the gel makes it easy to determine the sizes
of unknown DNAs.
-
+
DNA
migration
bromophenol blue
Note: bromophenol blue
migrates at
approximately the same
rate as a 300 bp DNA
molecule

29. Visualising the results
• The most common dye used to make DNA or RNA bands
visible for agarose gel electrophoresis is ethidium bromide,
usually abbreviated as EtBr. It fluoresces under UV light when
intercalated into DNA (or RNA). By running DNA through an
EtBr-treated gel and visualizing it with UV light. EtBr is a
known mutagen,But, safer alternatives are available.
Methylen Blue ,Bio-safe DNA stain etc.

30. Figure shows 1%Agarose gel electrophoresis of the PCR-amplified mecA methicillin
resistance gene. Lanes: 1: 50-bp ladder; 2: Negative control (S. aureus ATCC 8325-4);
3: Positive control (S. aureus strain COL); 4-7: S. aureus isolates showing 162
bpmecAamplicon
Result analysis and interpretation of Gel electrophoresis
BP DNA ladder _VE +VE S4 S5 S6 S7
Control